A lentivirally delivered photoactivatable GFP to assess continuity in the endoplasmic reticulum of neurones and glia.

Abstract

The endoplasmic reticulum (ER) is the largest intracellular membranous organelle. Functions of the ER are many and diverse, which include various biosynthetic, transport and signalling roles, central to cellular physiology, such as the biosynthesis of membrane and secretory proteins and the regulation of intracellular calcium. Its continuous lumen also serves as a highway for the distribution of proteins and ions to different regions of the cell, independent of the cytosol. The ER is an excitable organelle, capable of generating a regenerative wave of calcium release, which can propagate along the endomembrane throughout the entire cell, serving as a system of intracelluar communication in polarised cells. Nowhere is this feature of ER function more crucial than in neurones. The extremely polarised nature of nerve cells presents a unique challenge for the global co-ordination of localised physiological events such as growth cone guidance and synaptic plasticity. Clearly, the physical continuity of the neuronal ER lumen is central to its functionality as a conduit for communication. To further probe the continuity of ER in neurones and glia, we developed LV-PA-pIN-KDEL, a photoactivatable analogue of our recently described vector LV-pIN-KDEL, a lentivirally delivered ER-targeting soluble GFP. We demonstrate the ability of this vector to transduce astrocytes and neurones in culture and in cortical explants. Furthermore, we exploit the photoactivatable attributes of the vector together with a focal laser photoactivation protocol to reveal the continuous nature of the ER lumen in these cell types, presenting the first direct evidence of an astrocytic ER luminal continuum and providing more data to support the existence of a single ER lumen in neurones.